Ubiquitin-like conjugating protein

ABSTRACT

The invention provides a human ubiquitin-like conjugating protein (UBCLE) and polynucleotides which identify and encode UBCLE. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for treating or preventing disorders associated with expression of UBCLE.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of aubiquitin-like conjugating protein and to the use of these sequences inthe diagnosis, treatment, and prevention of cancer, and developmental,immune and neuronal disorders.

BACKGROUND OF THE INVENTION

The ubiquitin conjugation system (UCS) plays a major role in thedegradation of cellular proteins in eukaroytic cells and in somebacterial cells. UCS mediates the elimination of abnormal proteins andregulates the half-lives of other important regulatory proteins thatcontrol gene transcription and cell cycle progression. The UCS isreported to degrade mitotic cyclic kinases, oncoproteins, tumorsuppressors, viral proteins, transcriptional regulators, and receptorsassociated with signal transduction (Verma, R. et al. (1997) Science278:455-460; Ciechanover, A. (1994) Cell 79:13-21).

Several steps are involved in ubiquitin (Ub) conjugation and proteindegradation (Jentsch, S. (1992) Annu. Rev. Genet. 26:179-207). First,Ub, a small, heat stable protein, is activated by a ubiquitin-activatingenzyme (E1). This ATP dependent activation involves binding of theC-terminus of Ub to the thiol group of a cysteine residue of E1.Activated Ub is subsequently transferred to one of severalUb-conjugating enzymes (E2). Each E2 has a recognition subunit whichallows it to interact with proteins carrying a particular degradationsignal. E2 links the Ub molecule through its C-terminal glycine to aninternal lysine of the target protein. Different ubiquitin-dependentproteolytic pathways employ structurally similar, but distinct, E2s, andin some instances, accessory factors known as ubiquitin-ligases or E3s,are required to work in conjunction with E2s for recognition of certainsubstrates (Jensen, J. P. et al. (1995) J. Biol. Chem. 270:30408-30414).More than one Ub molecule may be needed to ubiquinate a target proteinwhich is subsequently recognized and degraded by a proteasome. Afterdegradation, Ub is released and reutilized.

Prior to activation, Ub is usually expressed as a fusion proteincomposed of an N-terminal ubiquitin and a C-terminal extension protein(CEP) or as a polyubiquitin protein with Ub monomers attached head totail. CEPs have characteristics of a variety of regulatory proteins;most are highly basic, contain up to 30% lysine and arginine residues,and have nucleic acid-binding domains (Monia, B. P. et al. (1989) J.Biol. Chem. 264:4093-4103). The fusion protein is an importantintermediate which appears to mediate co-regulation of the cell'stranslational and protein degradation activities, as well aslocalization of the inactive enzyme to specific cellular sites. Oncedelivered, C-terminal hydrolases cleave the fusion protein to release afunctional Ub (Monia et al., supra).

The E2s are important for substrate specificity in several UCS pathways.All E2s have a conserved ubiquitin conjugation (UBC) domain ofapproximately 16 kD, at least 35% identity with each other, and containa centrally located cysteine residue which is necessary forubiquitin-enzyme thiolester formation (Jentsch, supra). A highlyconserved proline-rich element is located N-terminal to the activecysteine residue. Structural variations outside of this conserved domainare used to separate the E2 enzymes into classes. The E2s of class 1(E2-1) consist of the conserved UBC domain and include yeast E2-1 andUBCs 4, 5, and 7. These E2s are thought to require E3 to carry out theiractivities (Jentsch, supra). UBC7 has been shown to recognize ubiquitinas a substrate and to form polyubiquitin chains in vitro (van Nocker, S.et al. (1996) J. Biol. Chem. 271:12150-58). E2s of class 2 (E2-2) havevarious unrelated C-terminal extensions that contribute to substratespecificity and cellular localization. The yeast E2-2 enzymes, UBC2 andUBC3, have highly acidic C-terminal extensions that promote interactionswith basic substrates such as histones. Yeast UBC6 has a hydrophobicsignal-anchor sequence that localizes the protein to the endoplasmicreticulum.

Abnormal activities of the UCS are implicated in a number of diseasesand disorders. These include, e.g., cachexia (Llovera, M. et al. (1995)Int. J. Cancer 61: 138-141), degradation of the tumor-suppressorprotein, p53 (Ciechanover, supra), and neurodegeneration such asobserved in Alzheimer's disease (Gregori, L. et al. (1994) Biochem.Biophys. Res. Commun. 203: 1731-1738). Since ubiquitin conjugation is arate-limiting step in antigen presentation, the ubiquitin degradationpathway may also have a critical role in the immune response (Grant E.P. et al. (1995) J. Immunol. 155: 3750-3758).

The discovery of new ubiquitin-conjugating-like protein and thepolynucleotides encoding it satisfies a need in the art by providing newcompositions which are useful in the diagnosis, treatment, andprevention of cancer, and developmental, immune and neuronal disorders.

SUMMARY OF THE INVENTION

The invention features a substantially purified polypeptide,ubiquitin-like conjugating protein (UBCLE), comprising the amino acidsequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.

The invention further provides a substantially purified variant of UBCLEhaving at least 90% amino acid identity to the amino acid sequence ofSEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also provides anisolated and purified polynucleotide sequence encoding the polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQID NO:1. The invention also includes an isolated and purifiedpolynucleotide variant having at least 90% polynucleotide identity tothe polynucleotide sequence encoding the polypeptide comprising theamino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.

Additionally, the invention provides a composition comprising apolynucleotide sequence encoding the polypeptide comprising the aminoacid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The inventionfurther provides an isolated and purified polynucleotide sequence whichhybridizes under stringent conditions to the polynucleotide sequenceencoding the polypeptide comprising the amino acid sequence of SEQ IDNO:1 or a fragment of SEQ ID NO:1, as well as an isolated and purifiedpolynucleotide sequence which is complementary to the polynucleotidesequence encoding the polypeptide comprising the amino acid sequence ofSEQ ID NO:1 or a fragment of SEQ ID NO:1.

The invention also provides an isolated and purified polynucleotidesequence comprising SEQ ID NO:2 or a fragment of SEQ ID NO:2, and anisolated and purified polynucleotide variant having at least 90%polynucleotide identity to the polynucleotide sequence comprising SEQ IDNO:2 or a fragment of SEQ ID NO:2. The invention also provides anisolated and purified polynucleotide sequence which is complementary tothe polynucleotide sequence comprising SEQ ID NO:2 or a fragment of SEQID NO:2.

The invention further provides an expression vector containing at leasta fragment of the polynucleotide sequence encoding the polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQID NO:1. In another aspect, the expression vector is contained within ahost cell.

The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQID NO:1, the method comprising the steps of: (a) culturing the host cellcontaining an expression vector containing at least a fragment of apolynucleotide sequence encoding UBCLE under conditions suitable for theexpression of the polypeptide; and (b) recovering the polypeptide fromthe host cell culture.

The invention also provides a pharmaceutical composition comprising asubstantially purified UBCLE having the amino acid sequence of SEQ IDNO:1 or a fragment of SEQ ID NO:1 in conjunction with a suitablepharmaceutical carrier.

The invention further includes a purified antibody which binds to apolypeptide comprising the amino acid sequence of SEQ ID NO:1 or afragment of SEQ ID NO:1, as well as a purified agonist and a purifiedantagonist of the polypeptide.

The invention also provides a method for treating or preventing acancer, the method comprising administering to a subject in need of suchtreatment an effective amount of an antagonist of UBCLE.

The invention also provides a method for treating or preventing andevelopmental disorder, the method comprising administering to a subjectin need of such treatment an effective amount of an antagonist of UBCLE.

The invention also provides a method for treating or preventing animmune disorder, the method comprising administering to a subject inneed of such treatment an effective amount of an antagonist of UBCLE.

The invention also provides a method for treating or preventing aneuronal disorder, the method comprising administering to a subject inneed of such treatment an effective amount of an antagonist of UBCLE.

The invention also provides a method for detecting a polynucleotideencoding UBCLE in a biological sample containing nucleic acids, themethod comprising the steps of:

(a) hybridizing the complement of the polynucleotide sequence encodingthe polypeptide comprising SEQ ID NO:1 or a fragment of SEQ ID NO:1 toat least one of the nucleic acids of the biological sample, therebyforming a hybridization complex; and (b) detecting the hybridizationcomplex, wherein the presence of the hybridization complex correlateswith the presence of a polynucleotide encoding UBCLE in the biologicalsample. In one aspect, the nucleic acids of the biological sample areamplified by the polymerase chain reaction prior to the hybridizingstep.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, and 1C show the amino acid sequence (SEQ ID NO:1) andnucleic acid sequence (SEQ ID NO:2) of UBCLE. The alignment was producedusing MACDNASIS PRO software (Hitachi Software Engineering Co. Ltd., SanBruno, Calif.).

FIG. 2 shows the amino acid sequence alignments between UBCLE (2501808;SEQ ID NO:1) and UBCH5C (GI 1145691; SEQ ID NO:3), produced using themultisequence alignment program of DNASTAR software (DNASTAR Inc,Madison Wis.).

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms "a," "an," and "the" include plural reference unless thecontext clearly dictates otherwise. Thus, for example, a reference to "ahost cell" includes a plurality of such host cells, and a reference to"an antibody" is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are cited for the purpose of describing and disclosing the celllines, vectors, and methodologies which are reported in the publicationsand which might be used in connection with the invention. Nothing hereinis to be construed as an admission that the invention is not entitled toantedate such disclosure by virtue of prior invention.

Definitions

"UBCLE," as used herein, refers to the amino acid sequences ofsubstantially purified UBCLE obtained from any species, particularly amammalian species, including bovine, ovine, porcine, murine, equine, andpreferably the human species, from any source, whether natural,synthetic, semi-synthetic, or recombinant.

The term "agonist," as used herein, refers to a molecule which, whenbound to UBCLE, increases or prolongs the duration of the effect ofUBCLE. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to and modulate the effect of UBCLE.

An "allele" or an "allelic sequence," as these terms are used herein, isan alternative form of the gene encoding UBCLE. Alleles may result fromat least one mutation in the nucleic acid sequence and may result inaltered mRNAs or in polypeptides whose structure or function may or maynot be altered. Any given natural or recombinant gene may have none,one, or many allelic forms. Common mutational changes which give rise toalleles are generally ascribed to natural deletions, additions, orsubstitutions of nucleotides. Each of these types of changes may occuralone, or in combination with the others, one or more times in a givensequence.

"Altered" nucleic acid sequences encoding UBCLE, as described herein,include those sequences with deletions, insertions, or substitutions ofdifferent nucleotides, resulting in a polynucleotide the same UBCLE or apolypeptide with at least one functional characteristic of UBCLE.Included within this definition are polymorphisms which may or may notbe readily detectable using a particular oligonucleotide probe of thepolynucleotide encoding UBCLE, and improper or unexpected hybridizationto alleles, with a locus other than the normal chromosomal locus for thepolynucleotide sequence encoding UBCLE. The encoded protein may also be"altered," and may contain deletions, insertions, or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent UBCLE. Deliberate amino acid substitutions maybe made on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues, as long as the biological or immunological activity of UBCLEis retained. For example, negatively charged amino acids may includeaspartic acid and glutamic acid, positively charged amino acids mayinclude lysine and arginine, and amino acids with uncharged polar headgroups having similar hydrophilicity values may include leucine,isoleucine, and valine; glycine and alanine; asparagine and glutamine;serine and threonine; and phenylalanine and tyrosine.

The terms "amino acid" or "amino acid sequence," as used herein, referto an oligopeptide, peptide, polypeptide, or protein sequence, or afragment of any of these, and to naturally occurring or syntheticmolecules. In this context, "fragments" refers to fragments of UBCLEwhich are preferably about 5 to about 15 amino acids in length and whichretain some biological activity or immunological activity of UBCLE.Where "amino acid sequence" is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, "amino acidsequence" and like terms are not meant to limit the amino acid sequenceto the complete native amino acid sequence associated with the recitedprotein molecule.

"Amplification," as used herein, relates to the production of additionalcopies of a nucleic acid sequence. Amplification is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart. (See, e.g., Dieffenbach, C. W. and G. S. Dveksler (1995) PCRPrimer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.)

The term "antagonist," as it is used herein, refers to a molecule which,when bound to UBCLE, decreases the amount or the duration of the effectof the biological or immunological activity of UBCLE. Antagonists mayinclude proteins, nucleic acids, carbohydrates, antibodies, or any othermolecules which decrease the effect of UBCLE.

As used herein, the term "antibody" refers to intact molecules as wellas to fragments thereof, such as Fab F(ab')₂, and Fv fragments, whichare capable of binding the epitopic determinant. Antibodies that bindUBCLE polypeptides can be prepared using intact polypeptides or usingfragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to immunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). The coupled peptide is then used to immunizethe animal.

The term "antigenic determinant," as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or a fragment of a protein is usedto immunize a host animal, numerous regions of the protein may inducethe production of antibodies which bind specifically to antigenicdeterminants (given regions or three-dimensional structures on theprotein). An antigenic determinant may compete with the intact antigen(i.e., the immunogen used to elicit the immune response) for binding toan antibody.

The term "antisense," as used herein, refers to any compositioncontaining a nucleic acid sequence which is complementary to a specificDNA or RNA sequence. The term "antisense strand" is used in reference toa nucleic acid strand that is complementary to the "sense" strand.Antisense molecules may be produced by any method including synthesis ortranscription. Once introduced into a cell, the complementarynucleotides combine with natural sequences produced by the cell to formduplexes and to block either transcription or translation. Thedesignation "negative" can refer to the antisense strand, and thedesignation "positive" can refer to the sense strand.

As used herein, the term "biologically active," refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, "immunologically active" refers to thecapability of the natural, recombinant, or synthetic UBCLE, or of anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The terms "complementary" or "complementarity," as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base pairing. For example, the sequence"A-G-T" binds to the complementary sequence "T-C-A." Complementaritybetween two single-stranded molecules may be "partial," such that onlysome of the nucleic acids bind, or it may be "complete," such that totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of the hybridization between the nucleicacid strands. This is of particular importance in amplificationreactions, which depend upon binding between nucleic acids strands, andin the design and use of peptide nucleic acid (PNA) molecules.

A "composition comprising a given polynucleotide sequence" or a"composition comprising a given amino acid sequence," as these terms areused herein, refer broadly to any composition containing the givenpolynucleotide or amino acid sequence. The composition may comprise adry formulation or an aqueous solution. Compositions comprisingpolynucleotide sequences encoding UBCLE or fragments of UBCLE may beemployed as hybridization probes. The probes may be stored infreeze-dried form and may be associated with a stabilizing agent such asa carbohydrate. In hybridizations, the probe may be deployed in anaqueous solution containing salts (e.g., NaCl), detergents (e.g., SDS),and other components (e.g., Denhardt's solution, dry milk, salmon spermDNA, etc.).

The phrase "consensus sequence," as used herein, refers to a nucleicacid sequence which has been resequenced to resolve uncalled bases,extended using XL-PCR Kit (Perkin Elmer, Norwalk, Conn.) in the 5'and/or the 3' direction, and resequenced, or which has been assembledfrom the overlapping sequences of more than one Incyte Clone using acomputer program for fragment assembly, such as the GELVIEW fragmentassembly system (GCG, Madison, Wis.). Some sequences have been bothextended and assembled to produce the consensus sequence.

As used herein, the term "correlates with expression of apolynucleotide" indicates that the detection of the presence of nucleicacids, the same or related to a nucleic acid sequence encoding UBCLE, bynorthern analysis is indicative of the presence of nucleic acidsencoding UBCLE in a sample, and thereby correlates with expression ofthe transcript from the polynucleotide encoding UBCLE.

A "deletion," as the term is used herein, refers to a change in theamino acid or nucleotide sequence that results in the absence of one ormore amino acid residues or nucleotides.

The term "derivative," as used herein, refers to the chemicalmodification of UBCLE, of a polynucleotide sequence encoding UBCLE, orof a polynucleotide sequence complementary to a polynucleotide sequenceencoding UBCLE. Chemical modifications of a polynucleotide sequence caninclude, for example, replacement of hydrogen by an alkyl, acyl, oramino group. A derivative polynucleotide encodes a polypeptide whichretains at least one biological or immunological function of the naturalmolecule. A derivative polypeptide is one modified by glycosylation,pegylation, or any similar process that retains a at least onebiological or immunological function of the polypeptide from which itwas derived.

The term "homology," as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology. Theword "identity" may substitute for the word "homology." A partiallycomplementary sequence that at least partially inhibits an identicalsequence from hybridizing to a target nucleic acid is referred to as"substantially homologous." The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization, and the like) under conditions of reduced stringency. Asubstantially homologous sequence or hybridization probe will competefor and inhibit the binding of a completely homologous sequence to thetarget sequence under conditions of reduced stringency. This is not tosay that conditions of reduced stringency are such that non-specificbinding is permitted, as reduced stringency conditions require that thebinding of two sequences to one another be a specific (i.e., aselective) interaction. The absence of non- specific binding may betested by the use of a second target sequence which lacks even a partialdegree of complementarity (e.g., less than about 30% homology oridentity). In the absence of non-specific binding, the substantiallyhomologous sequence or probe will not hybridize to the secondnon-complementary target sequence.

"Human artificial chromosomes" (HACs), as described herein, are linearmicrochromosomes which may contain DNA sequences of about 10 K to 10 Min size, and which contain all of the elements required for stablemitotic chromosome segregation and maintenance. (Harrington, J. J. etal. (1997) Nat Genet. 15:345-355.)

The term "humanized antibody," as used herein, refers to antibodymolecules in which the amino acid sequence in the non-antigen bindingregions has been altered so that the antibody more closely resembles ahuman antibody, and still retains its original binding ability.

"Hybridization," as the term is used herein, refers to any process bywhich a strand of nucleic acid binds with a complementary strand throughbase pairing.

As used herein, the term "hybridization complex" as used herein, refersto a complex formed between two nucleic acid sequences by virtue of theformation of hydrogen bonds between complementary bases. A hybridizationcomplex may be formed in solution (e.g., C₀ t or R₀ t analysis) orformed between one nucleic acid sequence present in solution and anothernucleic acid sequence immobilized on a solid support (e.g., paper,membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenfixed).

The words "insertion" or "addition," as used herein, refer to changes inan amino acid or nucleotide sequence resulting in the addition of one ormore amino acid residues or nucleotides, respectively, to the sequencefound in the naturally occurring molecule.

The term "microarray," as used herein, refers to an array of distinctpolynucleotides or oligonucleotides arrayed on a substrate, such aspaper, nylon or any other type of membrane, filter, chip, glass slide,or any other suitable solid support.

The term "modulate," as it appears herein, refers to a change in theactivity of UBCLE. For example, modulation may cause an increase or adecrease in protein activity, binding characteristics, or any otherbiological, functional, or immunological properties of UBCLE.

The phrases "nucleic acid" or "nucleic acid sequence," as used herein,refer to an oligonucleotide, nucleotide, polynucleotide, or any fragmentthereof, to DNA or RNA of genomic or synthetic origin which may besingle-stranded or double-stranded and may represent the sense or theantisense strand, to peptide nucleic acid (PNA), or to any DNA-like orRNA-like material. In this context, "fragments" refers to those nucleicacid sequences which are greater than about 60 nucleotides in length,and most preferably are at least about 100 nucleotides, at least about1000 nucleotides, or at least about 10,000 nucleotides in length.

The term "oligonucleotide," as used herein, refers to a nucleic acidsequence of at least about 6 nucleotides to 60 nucleotides, preferablyabout 15 to 30 nucleotides, and most preferably about 20 to 25nucleotides, which can be used in PCR amplification or in ahybridization assay or microarray. As used herein, the term"oligonucleotide" is substantially equivalent to the terms "amplimers,""primers," "oligomers," and "probes," as these terms are commonlydefined in the art.

"Peptide nucleic acid" (PNA), as used herein, refers to an antisensemolecule or anti-gene agent which comprises an oligonucleotide of atleast about 5 nucleotides in length linked to a peptide backbone ofamino acid residues ending in lysine. The terminal lysine conferssolubility to the composition. PNAs preferentially bind complementarysingle stranded DNA and RNA and stop transcript elongation, and may bepegylated to extend their lifespan in the cell. (Nielsen, P. E. et al.(1993) Anticancer Drug Des. 8:53-63.

The term "sample," as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acids encoding UBCLE,or fragments thereof, or UBCLE itself may comprise a bodily fluid; anextract from a cell, chromosome, organelle, or membrane isolated from acell; a cell; genomic DNA, RNA, or cDNA (in solution or bound to a solidsupport); a tissue; a tissue print; and the like.

As used herein, the terms "specific binding" or "specifically binding"refer to that interaction between a protein or peptide and an agonist,an antibody, or an antagonist. The interaction is dependent upon thepresence of a particular structure of the protein recognized by thebinding molecule (i.e., the antigenic determinant or epitope). Forexample, if an antibody is specific for epitope "A," the presence of apolypeptide containing the epitope A, or the presence of free unlabeledA, in a reaction containing free labeled A and the antibody will reducethe amount of labeled A that binds to the antibody.

As used herein, the term "stringent conditions" refers to conditionswhich permit hybridization between polynucleotide sequences and theclaimed polynucleotide sequences. Suitably stringent conditions can bedefined by, for example, the concentrations of salt or formamide in theprehybridization and hybridization solutions, or by the hybridizationtemperature, and are well known in the art. In particular, stringencycan be increased by reducing the concentration of salt, increasing theconcentration of formamide, or raising the hybridization temperature.

For example, hybridization under high stringency conditions could occurin about 50% formamide at about 37° C. to 42° C. Hybridization couldoccur under reduced stringency conditions in about 35% to 25% formamideat about 30° C. to 35° C. In particular, hybridization could occur underhigh stringency conditions at 42° C. in 50% formamide, 5×SSPE, 0.3% SDS,and 200 μg/ml sheared and denatured salmon sperm DNA. Hybridizationcould occur under reduced stringency conditions as described above, butin 35% formamide at a reduced temperature of 35° C. The temperaturerange corresponding to a particular level of stringency can be furthernarrowed by calculating the purine to pyrimidine ratio of the nucleicacid of interest and adjusting the temperature accordingly. Variationson the above ranges and conditions are well known in the art.

The term "substantially purified," as used herein, refers to nucleicacid or amino acid sequences that are removed from their naturalenvironment and are isolated or separated, and are at least about 60%free, preferably about 75% free, and most preferably about 90% free fromother components with which they are naturally associated.

A "substitution," as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

"Transformation," as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. Transformation mayoccur under natural or artificial conditions according to variousmethods well known in the art, and may rely on any known method for theinsertion of foreign nucleic acid sequences into a prokaryotic oreukaryotic host cell. The method for transformation is selected based onthe type of host cell being transformed and may include, but is notlimited to, viral infection, electroporation, heat shock, lipofection,and particle bombardment. The term "transformed" cells includes stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome, and refers to cells which transiently express the insertedDNA or RNA for limited periods of time.

A "variant" of UBCLE, as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have"conservative" changes, wherein a substituted amino acid has similarstructural or chemical properties (e.g., replacement of leucine withisoleucine). More rarely, a variant may have "nonconservative" changes(e.g., replacement of glycine with tryptophan). Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

The Invention

The invention is based on the discovery of a new human ubiquitin-likeconjugating protein (UBCLE), the polynucleotides encoding UBCLE, and theuse of these compositions for the diagnosis, treatment, or prevention ofcancer, and developmental, immune, and neuronal disorders.

Nucleic acids encoding the UBCLE of the present invention were firstidentified in Incyte Clone 2501808 from the adrenal tumor cDNA library(ADRETUT05) using a computer search for amino acid sequence alignments.A consensus sequence, SEQ ID NO:2, was derived from the followingoverlapping and/or extended nucleic acid sequences: Incyte Clones2501808 (ADRETUT05), 291812 (TMLR3DT01), and 743853 (BRAITUT01).

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO:1. UBCLE is 198 amino acids inlength and has a potential ubiquitin conjugating enzyme active sitebetween residues Y₇₅ and V₈₉, a potential N-glycosylation site atresidue N₁₇₆, a potential cAMP phosphorylation site at residue R₁₈₁, apotential casein kinase II phosphorylation site at residue T₄₄, and twopotential protein kinase C phosphorylation sites at residues T₁₄₇ andT₁₇₈. As shown in FIG. 2, UBCLE has chemical and structural homologywith UBCH5C (GI 1145691; SEQ ID NO:3). In particular, UBCLE and UBCH5Cshare 32% sequence identity. Northern analysis shows the expression ofthis sequence in various cDNA libraries, at least 52% of which areimmortalized or cancerous, at least 39% of which involve cellproliferation. Of particular note is the expression in nervous systemtissues.

The invention also encompasses UBCLE variants. A preferred UBCLE variantis one having at least about 80%, more preferably at least about 90%,and most preferably at least about 95% amino acid sequence identity tothe UBCLE amino acid sequence.

The invention also encompasses polynucleotides which encode UBCLE. In aparticular embodiment, the invention encompasses a polynucleotidesequence comprising the sequence of SEQ ID NO:2, which encodes an UBCLE.

The invention also encompasses a variant of a polynucleotide sequenceencoding UBCLE. In particular, such a variant polynucleotide sequencewill have at least about 80%, more preferably at least about 90%, andmost preferably at least about 95% polynucleotide sequence identity tothe polynucleotide sequence encoding UBCLE. A particular aspect of theinvention encompasses a variant of SEQ ID NO:2 which has at least about80%, more preferably at least about 90%, and most preferably at leastabout 95% polynucleotide sequence identity to SEQ ID NO:2.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of polynucleotidesequences encoding UBCLE, some bearing minimal homology to thepolynucleotide sequences of any known and naturally occurring gene, maybe produced. Thus, the invention contemplates each and every possiblevariation of polynucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe polynucleotide sequence of naturally occurring UBCLE, and all suchvariations are to be considered as being specifically disclosed.

Although nucleotide sequences which encode UBCLE and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring UBCLE under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding UBCLE or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding UBCLE and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

The invention also encompasses production of DNA sequences which encodeUBCLE and UBCLE derivatives, or fragments thereof, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents that are well known in the art. Moreover, synthetic chemistrymay be used to introduce mutations into a sequence encoding UBCLE or anyfragment thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed polynucleotide sequences, and, inparticular, to those shown in SEQ ID NO:2, or a fragment of SEQ ID NO:2,under various conditions of stringency as taught in Wahl, G. M. and S.L. Berger (1987; Methods Enzymol. 152:399-407) and Kimmel, A. R. (1987;Methods Enzymol. 152:507-511).

Methods for DNA sequencing are well known and generally available in theart and may be used to practice any of the embodiments of the invention.The methods may employ such enzymes as the Klenow fragment of DNApolymerase I, SEQUENASE (US Biochemical Corp., Cleveland, Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, ll.), or combinations of polymerases and proofreadingexonucleases such as those found in the amplification system (GIBCO/BRLGaithersburg, Md.). Preferably, the process is automated with machinessuch as the Hamilton MICRO LAB 2200 (Hamilton, Reno, Nev.), Peltierthermal cycler (PTC200; MJ Research, Watertown, Mass.) and the ABICATALYST and 373 and 377 DNA SEQUENCERS (Perkin Elmer).

The nucleic acid sequences encoding UBCLE may be extended utilizing apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences, such as promoters and regulatoryelements. For example, one method which may be employed,restriction-site PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus. (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322.) In particular, genomic DNA is first amplified in thepresence of a primer to a linker sequence and a primer specific to theknown region. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region. (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186.) The primers may be designed usingcommercially available software such as OLIGO 4.06 primer analysissoftware (National Biosciences Inc., Plymouth, Minn.) or anotherappropriate program to be about 22 to 30 nucleotides in length, to havea GC content of about 50% or more, and to anneal to the target sequenceat temperatures of about 68° C. to 72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

Another method which may be used is capture PCR, which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA. (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119.) In this method, multiple restriction enzymedigestions and ligations may be used to place an engineereddouble-stranded sequence into an unknown fragment of the DNA moleculebefore performing PCR. Another method which may be used to retrieveunknown sequences is that of Parker, J. D. et al. (1991; Nucleic AcidsRes. 19:3055-3060). Additionally, one may use PCR, nested primers, andPROMOTER FINDER libraries to walk genomic DNA (Clontech, Palo Alto,Calif.). This process avoids the need to screen libraries and is usefulin finding intron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable in that they will include moresequences which contain the 5' regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into 5' non-transcribedregulatory regions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and a charge coupled device camera for detection of the emittedwavelengths. Output/light intensity may be converted to electricalsignal using appropriate software (e.g., SEQUENCE NAVIGATOR andGENOTYPER, Perkin Elmer), and the entire process from loading of samplesto computer analysis and electronic data display may be computercontrolled. Capillary electrophoresis is especially preferable for thesequencing of small pieces of DNA which might be present in limitedamounts in a particular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode UBCLE may be used in recombinant DNAmolecules to direct expression of UBCLE, or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced, and these sequences may be used to clone and expressUBCLE.

As will be understood by those of skill in the art, it may beadvantageous to produce UBCLE-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce an RNA transcript havingdesirable properties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter UBCLEencoding sequences for a variety of reasons including, but not limitedto, alterations which modify the cloning, processing, and/or expressionof the gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding UBCLE may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of UBCLE activity, it may be useful toencode a chimeric UBCLE protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the UBCLE encoding sequence and theheterologous protein sequence, so that UBCLE may be cleaved and purifiedaway from the heterologous moiety.

In another embodiment, sequences encoding UBCLE may be synthesized, inwhole or in part, using chemical methods well known in the art. (SeeCaruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 7:215-223,and Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 7:225-232.)Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of UBCLE, or a fragment thereof.For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge, J. Y. et al. (1995) Science269:202-204) and automated synthesis may be achieved using the ABI 431APeptide Synthesizer (Perkin Elmer).

The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography. (See, for example,Creighton, T. (1983) Proteins, Structures and Molecular Principles, WHFreeman and Co., New York, N.Y.) The composition of the syntheticpeptides may be confirmed by amino acid analysis or by sequencing. (See,for example, the Edman degradation procedure described in Creighton,supra.) Additionally, the amino acid sequence of UBCLE, or any partthereof, may be altered during direct synthesis and/or combined withsequences from other proteins, or any part thereof, to produce a variantpolypeptide.

In order to express a biologically active UBCLE, the nucleotidesequences encoding UBCLE or derivatives thereof may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding UBCLE andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989; Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y.) and Ausubel, F. M. etal. (1989; Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.).

A variety of expression vector/host systems may be utilized to containand express sequences encoding UBCLE. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus (CaMV) or tobacco mosaic virus (TMV)) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

The "control elements" or "regulatory sequences" are thosenon-translated regions of the vector (i.e., enhancers, promoters, and 5'and 3' untranslated regions) which interact with host cellular proteinsto carry out transcription and translation. Such elements may vary intheir strength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or PSPORT1 plasmid (GIBCO/BRL), and the like, may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO, and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding UBCLE,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for UBCLE. For example, when largequantities of UBCLE are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be used. Such vectors include, but are not limited to,multifunctional E. coli cloning and expression vectors such as theBLUESCRIPT phagemid (Stratagene), in which the sequence encoding UBCLEmay be ligated into the vector in frame with sequences for theamino-terminal Met and the subsequent 7 residues of β-galactosidase sothat a hybrid protein is produced, pIN vectors (Van Heeke, G. and S. M.Schuster (1989) J. Biol. Chem. 264:5503-5509), and the like. PGEXvectors (Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

In the yeast Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters, such as alpha factor, alcoholoxidase, and PGH, may be used. For reviews, see Ausubel (supra) andGrant et al. (1987; Methods Enzymol. 153:516-544).

In cases where plant expression vectors are used, the expression ofsequences encoding UBCLE may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMVmay be used alone or in combination with the omega leader sequence fromTMV. (Takamatsu, N. (1987) EMBO J. 6:307-31 1.) Alternatively, plantpromoters such as the small subunit of RUBISCO or heat shock promotersmay be used. (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R.et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) ResultsProbl. Cell Differ. 17:85-105.) These constructs can be introduced intoplant cells by direct DNA transformation or pathogen-mediatedtransfection. Such techniques are described in a number of generallyavailable reviews. (See, for example, Hobbs, S. or Murry, L. E. inMcGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, NewYork, N.Y.; pp. 191-196.)

An insect system may also be used to express UBCLE. For example, in onesuch system, Autographa californica nuclear polyhedrosis virus (AcNPV)is used as a vector to express foreign genes in Spodoptera frugiperdacells or in Trichoplusia larvae. The sequences encoding UBCLE may becloned into a non-essential region of the virus, such as the polyhedringene, and placed under control of the polyhedrin promoter. Successfulinsertion of UBCLE will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which UBCLE may be expressed. (Engelhard, E.K. et al. (1994)Proc. Nat. Acad. Sci. 91:3224-3227.) In mammalian host cells, a numberof viral-based expression systems may be utilized. In cases where anadenovirus is used as an expression vector, sequences encoding UBCLE maybe ligated into an adenovirus transcription/translation complexconsisting of the late promoter and tripartite leader sequence.Insertion in a non-essential E1 or E3 region of the viral genome may beused to obtain a viable virus which is capable of expressing UBCLE ininfected host cells. (Logan, J. and T. Shenk (1984) Proc. Natl. Acad.Sci. 81:3655-3659.) In addition, transcription enhancers, such as theRous sarcoma virus (RSV) enhancer, may be used to increase expression inmammalian host cells.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained and expressed in aplasmid. HACs of about 6 M to 10 M are constructed and delivered viaconventional delivery methods (liposomes, polycationic amino polymers,or vesicles) for therapeutic purposes.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding UBCLE. Such signals include the ATGinitiation codon and adjacent sequences. In cases where sequencesencoding UBCLE and its initiation codon and upstream sequences areinserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a fragment thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers appropriate for the particularcell system used, such as those described in the literature. (Scharf, D.et al. (1994) Results Probl. Cell Differ. 20:125-162.)

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a "prepro" form of theprotein may also be used to facilitate correct insertion, folding,and/or function. Different host cells which have specific cellularmachinery and characteristic mechanisms for post-translationalactivities (e.g., CHO, HeLa, MDCK, HEK293, and W138), are available fromthe American Type Culture Collection (ATCC, Bethesda, Md.) and may bechosen to ensure the correct modification and processing of the foreignprotein.

For long term, high yield production of recombinant proteins, stableexpression is preferred. For example, cell lines capable of stablyexpressing UBCLE can be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for about 1 to 2 days in enriched media before being switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase genes (Wigler, M. et al. (1977) Cell 11:223-32) andadenine phosphoribosyltransferase genes (Lowy, I. et al. (1980) Cell22:817-23), which can be employed in tk⁻ or apr⁻ cells, respectively.Also, antimetabolite, antibiotic, or herbicide resistance can be used asthe basis for selection. For example, dhfr confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt confers resistance to the aminoglycosides neomycin andG-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14); and alsor pat confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murry, supra). Additional selectablegenes have been described, for example, trpB, which allows cells toutilize indole in place of tryptophan, or hisD, which allows cells toutilize histinol in place of histidine. (Hartman, S. C. and R. C.Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51.) Recently, the use ofvisible markers has gained popularity with such markers as anthocyanins,β-glucuronidase and its substrate GUS, and luciferase and its substrateluciferin. These markers can be used not only to identify transformants,but also to quantify the amount of transient or stable proteinexpression attributable to a specific vector system. (Rhodes, C. A. etal. (1995) Methods Mol. Biol. 55:121-131.)

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, the presence and expression of thegene may need to be confirmed. For example, if the sequence encodingUBCLE is inserted within a marker gene sequence, transformed cellscontaining sequences encoding UBCLE can be identified by the absence ofmarker gene function. Alternatively, a marker gene can be placed intandem with a sequence encoding UBCLE under the control of a singlepromoter. Expression of the marker gene in response to induction orselection usually indicates expression of the tandem gene as well.

Alternatively, host cells which contain the nucleic acid sequenceencoding UBCLE and express UBCLE may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein sequences.

The presence of polynucleotide sequences encoding UBCLE can be detectedby DNA-DNA or DNA-RNA hybridization or amplification using probes orfragments or fragments of polynucleotides encoding UBCLE. Nucleic acidamplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding UBCLE to detect transformantscontaining DNA or RNA encoding UBCLE.

A variety of protocols for detecting and measuring the expression ofUBCLE, using either polyclonal or monoclonal antibodies specific for theprotein, are known in the art. Examples of such techniques includeenzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),and fluorescence activated cell sorting (FACS). A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes on UBCLE is preferred, but a competitivebinding assay may be employed. These and other assays are well describedin the art, for example, in Hampton, R. et al. (1990; SerologicalMethods a Laboratory Manual, APS Press, St Paul, Minn.) and in Maddox,D. E. et al. (1983; J. Exp. Med. 158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding UBCLE includeoligolabeling, nick translation, end-labeling, or PCR amplificationusing a labeled nucleotide. Alternatively, the sequences encoding UBCLE,or any fragments thereof, may be cloned into a vector for the productionof an mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits, such as those provided by Pharmacia &Upjohn (Kalamazoo, Mich.), Promega (Madison, Wis.), and U.S. BiochemicalCorp. (Cleveland, Ohio). Suitable reporter molecules or labels which maybe used for ease of detection include radionuclides, enzymes,fluorescent, chemiluminescent, or chromogenic agents, as well assubstrates, cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding UBCLE may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeUBCLE may be designed to contain signal sequences which direct secretionof UBCLE through a prokaryotic or eukaryotic cell membrane. Otherconstructions may be used to join sequences encoding UBCLE to nucleotidesequences encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences, such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.), between the purificationdomain and the UBCLE encoding sequence may be used to facilitatepurification. One such expression vector provides for expression of afusion protein containing UBCLE and a nucleic acid encoding 6 histidineresidues preceding a thioredoxin or an enterokinase cleavage site. Thehistidine residues facilitate purification on immobilized metal ionaffinity chromatography (IMIAC; described in Porath, J. et al. (1992)Prot. Exp. Purif. 3: 263-281)), while the enterokinase cleavage siteprovides a means for purifying UBCLE from the fusion protein. Adiscussion of vectors which contain fusion proteins is provided inKroll, D. J. et al. (1993; DNA Cell Biol. 12:441-453).

Fragments of UBCLE may be produced not only by recombinant production,but also by direct peptide synthesis using solid-phase techniques.(Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154.) Protein synthesismay be performed by manual techniques or by automation. Automatedsynthesis may be achieved, for example, using the Applied Biosystems431A PEPTIDE SYNTHESIZER (Perkin Elmer). Various fragments of UBCLE maybe synthesized separately and then combined to produce the full lengthmolecule.

Therapeutics

Chemical and structural homology exists between UBCLE and human UBC115C(GI 1145690). In addition, UBCLE is expressed in cancer, anddevelopmental, immune, and neuronal disorders where UBCLE plays a rolein the cell cycle and in cell signaling. Therefore, UBCLE appears toplay a role in cancer, and developmental, immune, and neuronaldisorders.

Degradation of tumor suppressor proteins such as p53 by E2 enzymes maycontribute to the development of cancer. Therefore, in one embodiment,an antagonist of UBCLE may be administered to a subject to treat orprevent a cancer. Such cancers can include, but are not limited to,adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus.

In an additional embodiment, a vector expressing the complement of thepolynucleotide encoding UBCLE may be administered to a subject to treator prevent a cancer including, but not limited to, those describedabove.

Abnormalities in processing of neural proteins (AP) by enzymes of theUCS may contribute to neuronal disorders. Therefore, in anotherembodiment, an antagonist which modulates the activity of UBCLE may bcadministered to a subject to treat or prevent a neuronal disorder. Suchneuronal disorders can include, but are not limited to, akathesia,Alzheimer's disease, amnesia, amyotrophic lateral sclerosis, bipolardisorder, catatonia, cerebral neoplasms, dementia, depression, Down'ssyndrome, tardive dyskinesia, dystonias, epilepsy, Huntington's disease,multiple sclerosis, Parkinson's disease, paranoid psychoses,schizophrenia, and Tourette's disorder.

In an additional embodiment, a vector expressing the complement of thepolynucleotide encoding UBCLE may be administered to a subject to treator prevent a neuronal disorder including, but not limited to, thosedisorders described above.

In a further embodiment, an antagonist of UBCLE may be administered to asubject to treat or prevent a developmental disorder. Such developmentaldisorders may include, but are not limited to, renal tubular acidosis,Cushing's syndrome, achondroplastic dwarfism, Duchenne and Beckermuscular dystrophy, gonadal dysgenesis, myelodysplastic syndrome,hereditary mucoepithelial dysplasia, hereditary keratodermas, hereditaryneuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis,hypothyroidism, hydrocephalus, seizure disorders such as Syndenham'schorea and cerebral palsy, spinal bifida, and congenital glaucoma,cataract, or sensorineural hearing loss.

In an additional embodiment, a vector expressing the complement of thepolynucleotide encoding UBCLE may be administered to a subject to treator prevent a developmental disorder including, but not limited to, thosedisorders described above.

In a further embodiment, an antagonist of UBCLE may be administered to asubject to treat or prevent an immune disorder. Such immune disordersmay include, but are not limited to, Addison's disease, adultrespiratory distress syndrome, allergies, ankylosing spondylitis,amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolyticanemia, autoimmune thyroiditis, bronchitis, cholecystitis, contactdermayitis, Crohn's disease, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, erythema nodosum, atrophic gastritis,glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome,lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardialor pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjogren's syndrome,systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis,ulcerative colitis, Werner syndrome, and complications of cancer,hemodialysis, and extracorporeal circulation; viral, bacterial, fungal,parasitic, protozoal, and helminthic infections; and trauma.

In an additional embodiment, a vector expressing the complement of thepolynucleotide encoding UBCLE may be administered to a subject to treator prevent an immune disorder including, but not limited to, thosedisorders described above.

In one aspect, an antibody which specifically binds UBCLE may be useddirectly as an antagonist or indirectly as a targeting or deliverymechanism for bringing a pharmaceutical agent to cells or tissue whichexpress UBCLE.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences, or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of UBCLE may be produced using methods which are generallyknown in the art. In particular, purified UBCLE may be used to produceantibodies or to screen libraries of pharmaceutical agents to identifythose which specifically bind UBCLE. Antibodies to UBCLE may also begenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric,and single chain antibodies, Fab fragments, and fragments produced by aFab expression library. Neutralizing antibodies (i.e., those whichinhibit dimer formation) are especially preferred for therapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others may be immunized by injectionwith UBCLE or with any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to UBCLE have an amino acid sequence consisting of atleast about 5 amino acids, and, more preferably, of at least about 10amino acids. It is also preferable that these oligopeptides, peptides,or fragments are identical to a portion of the amino acid sequence ofthe natural protein and contain the entire amino acid sequence of asmall, naturally occurring molecule. Short stretches of UBCLE aminoacids may be fused with those of another protein, such as KLH, andantibodies to the chimeric molecule may be produced.

Monoclonal antibodies to UBCLE may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique. (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. etal. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc.Natl.

Acad. Sci. 80:2026-2030; and Cole, S. P. et al. (1984) Mol. Cell Biol.62:109-120.)

In addition, techniques developed for the production of "chimericantibodies," such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (Morrison, S. L. et al. (1984)Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984)Nature 312:604-608; and Takeda, S. et al. (1985) Nature 314:452-454.)Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceUBCLE-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries.(Burton D. R. (1991) Proc. Natl. Acad. Sci. 88:11120-11123.)

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature.(Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837, andWinter, G. et al. (1991) Nature 349:293-299.)

Antibody fragments which contain specific binding sites for UBCLE mayalso be generated. For example, such fragments include, but are notlimited to, F(ab')2 fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab')2 fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity. (Huse, W. D. etal. (1989) Science 254:1275-1281.) Various immunoassays may be used forscreening to identify antibodies having the desired specificity.Numerous protocols for competitive binding or immunoradiometric assaysusing either polyclonal or monoclonal antibodies with establishedspecificities are well known in the art. Such immunoassays typicallyinvolve the measurement of complex formation between UBCLE and itsspecific antibody. A two-site, monoclonal-based immunoassay utilizingmonoclonal antibodies reactive to two non-interfering UBCLE epitopes ispreferred, but a competitive binding assay may also be employed.(Maddox, supra.)

In another embodiment of the invention, the polynucleotides encodingUBCLE, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, the complement of thepolynucleotide encoding UBCLE may be used in situations in which itwould be desirable to block the transcription of the mRNA. Inparticular, cells may be transformed with sequences complementary topolynucleotides encoding UBCLE. Thus, complementary molecules orfragments may be used to modulate UBCLE activity, or to achieveregulation of gene function. Such technology is now well known in theart, and sense or antisense oligonucleotides or larger fragments can bedesigned from various locations along the coding or control regions ofsequences encoding UBCLE.

Expression vectors derived from retroviruses, adenoviruses, or herpes orvaccinia viruses, or from various bacterial plasmids, may be used fordelivery of nucleotide sequences to the targeted organ, tissue, or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors which will express nucleic acid sequencecomplementary to the polynucleotides of the gene encoding UBCLE. Thesetechniques are described, for example, in Sambrook (supra) and inAusubel (supra).

Genes encoding UBCLE can be turned off by transforming a cell or tissuewith expression vectors which express high levels of a polynucleotide orfragment thereof encoding UBCLE. Such constructs may be used tointroduce untranslatable sense or antisense sequences into a cell. Evenin the absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector, and may last even longer if appropriatereplication elements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning complementary sequences or antisense molecules (DNA, RNA, orPNA) to the control, 5', or regulatory regions of the gene encodingUBCLE. Oligonucleotides derived from the transcription initiation site,e.g., between about positions -10 and +10 from the start site, arepreferred. Similarly, inhibition can be achieved using triple helixbase-pairing methodology. Triple helix pairing is useful because itcauses inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature. (Gee, J. E. et al. (1994) in Huber, B.E. and B. I. Carr, Molecular and immunologic Approaches, FuturaPublishing Co., Mt. Kisco, N.Y.) A complementary sequence or antisensemolecule may also be designed to block translation of mRNA by preventingthe transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules specifically andefficiently catalyze endonucleolytic cleavage of sequences encodingUBCLE.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding UBCLE. SuchDNA sequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize complementary RNA constitutivelyor inducibly can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5' and/or 3' ends of the moleculeor the use of phosphorothioate or 2' O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections, or bypolycationic amino polymers may be achieved using methods which are wellknown in the art, such as those described in Goldman, C. K. et al.(1997; Nature Biotechnology 15:462-466).

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of UBCLE, antibodies toUBCLE, and mimetics, agonists, antagonists, or inhibitors of UBCLE. Thecompositions may be administered alone or in combination with at leastone other agent, such as a stabilizing compound, which may beadministered in any sterile, biocompatible pharmaceutical carrierincluding, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with fillers or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils, such as sesame oil, or synthetic fatty acid esters, such asethyl oleate, triglycerides, or liposomes. Non-lipid polycationic aminopolymers may also be used for delivery. Optionally, the suspension mayalso contain suitable stabilizers or agents to increase the solubilityof the compounds and allow for the preparation of highly concentratedsolutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tendto be more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7%mannitol, at a pH range of 4.5 to 5.5, that is combined with bufferprior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of UBCLE, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays of neoplastic cells, forexample, or in animal models, usually mice, rabbits, dogs, or pigs. Ananimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example UBCLE or fragments thereof, antibodies of UBCLE,and agonists, antagonists or inhibitors of UBCLE, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED50 (the dosetherapeutically effective in 50% of the population) or LD50 (the doselethal to 50% of the population) statistics. The dose ratio betweentherapeutic and toxic effects is the therapeutic index, and it can beexpressed as the LD50/ED50 ratio. Pharmaceutical compositions whichexhibit large therapeutic indices are preferred. The data obtained fromcell culture assays and animal studies is used in formulating a range ofdosage for human use. The dosage contained in such compositions ispreferably within a range of circulating concentrations that include theED50 with little or no toxicity. The dosage varies within this rangedepending upon the dosage form employed, the sensitivity of the patient,and the route of administration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, diet,time and frequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on the half-life and clearancerate of the particular formulation.

Normal dosage amounts may vary from 0.1 μg to 100,000 μg, up to a totaldose of about 1 gram, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

Diagnostics

In another embodiment, antibodies which specifically bind UBCLE may beused for the diagnosis of disorders characterized by expression ofUBCLE, or in assays to monitor patients being treated with UBCLE oragonists, antagonists, and inhibitors of UBCLE. Antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for UBCLE includemethods which utilize the antibody and a label to detect UBCLE in humanbody fluids or in extracts of cells or tissues. The antibodies may beused with or without modification, and may be labeled by covalent ornon-covalent joining with a reporter molecule. A wide variety ofreporter molecules, several of which are described above, are known inthe art and may be used.

A variety of protocols for measuring UBCLE, including ELISAs, RIAs, andFACS, are known in the art and provide a basis for diagnosing altered orabnormal levels of UBCLE expression. Normal or standard values for UBCLEexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toUBCLE under conditions suitable for complex formation The amount ofstandard complex formation may be quantified by various methods,preferably by photometric means. Quantities of UBCLE expressed insubject, control, and disease samples from biopsied tissues are comparedwith the standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingUBCLE may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofUBCLE may be correlated with disease. The diagnostic assay may be usedto distinguish between absence, presence, and excess expression ofUBCLE, and to monitor regulation of UBCLE levels during therapeuticintervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding UBCLE or closely related molecules may be used to identifynucleic acid sequences which encode UBCLE. The specificity of the probe,whether it is made from a highly specific region (e.g., the 5'regulatory region) or from a less specific region (e.g., the 3' codingregion), and the stringency of the hybridization or amplification(maximal, high, intermediate, or low), will determine whether the probeidentifies only naturally occurring sequences encoding UBCLE, alleles,or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50% of the nucleotides from any ofthe UBCLE encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and may be derived from the sequence of SEQID NO:2 or from genomic sequences including promoter and enhancerelements and introns of the naturally occurring UBCLE.

Means for producing specific hybridization probes for DNAs encodingUBCLE include the cloning of polynucleotide sequences encoding UBCLE orUBCLE derivatives into vectors for the production of mRNA probes. Suchvectors are known in the art, are commercially available, and may beused to synthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, by radionuclides such as ³² P or ³⁵ S, or by enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems, and the like.

Polynucleotide sequences encoding UBCLE may be used for the diagnosis ofa disorder associated with expression of UBCLE. Disorders include, butare not limited to, cancers such as adenocarcinoma, leukemia, lymphoma,melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancersof the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix,gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver,lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivaryglands, skin, spleen, testis, thymus, thyroid, and uterus; neuronaldisorders such as akathesia, Alzheimer's disease, amnesia, amyotrophiclateral sclerosis, bipolar disorder, catatonia, cerebral neoplasms,dementia, depression, Down's syndrome, tardive dyskinesia, dystonias,epilepsy, Huntington's disease, multiple sclerosis, Parkinson's disease,paranoid psychoses, schizophrenia, and Tourette's disorder;develpomental disorders such as renal tubular acidosis, Cushing'ssyndrome, achondroplastic dwarfism, Duchenne and Becker musculardystrophy, gonadal dysgenesis, myelodysplastic syndrome, hereditarymucoepithelial dysplasia, hereditary keratodermas, hereditaryneuropathies such as Charcot-Marie-Tooth disease and neurofibromatosis,hypothyroidism, hydrocephalus, seizure disorders such as Syndenham'schorea and cerebral palsy, spinal bifida, and congenital glaucoma,cataract, or sensorineural hearing loss; and immune disorders such asAddison's disease, adult respiratory distress syndrome, allergies,ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis,autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis,cholecystitis, contact dermayitis, Crohn's disease, atopic dermatitis,dermatomyositis, diabetes mellitus, emphysema, erythema nodosum,atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout,Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritablebowel syndrome, lupus erythematosus, multiple sclerosis, myastheniagravis, myocardial or pericardial inflammation, osteoarthritis,osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis,scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupuserythematosus, systemic sclerosis, ulcerative colitis, Werner syndrome,and complications of cancer, hemodialysis, and extracorporealcirculation; viral, bacterial, fungal, parasitic, protozoal, andhelminthic infections; and trauma. The polynucleotide sequences encodingUBCLE may be used in Southern or northern analysis, dot blot, or othermembrane-based technologies; in PCR technologies; in dipstick, pin, andELISA assays; and in microarrays utilizing fluids or tissues frompatient biopsies to detect altered UBCLE expression. Such qualitative orquantitative methods are well known in the art.

In a particular aspect, the nucleotide sequences encoding UBCLE may beuseful in assays that detect the presence of associated disorders,particularly those mentioned above. The nucleotide sequences encodingUBCLE may be labeled by standard methods and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the patient sample is significantlyaltered from that of a comparable control sample, the nucleotidesequences have hybridized with nucleotide sequences in the sample, andthe presence of altered levels of nucleotide sequences encoding UBCLE inthe sample indicates the presence of the associated disorder. Suchassays may also be used to evaluate the efficacy of a particulartherapeutic treatment regimen in animal studies, in clinical trials, orin monitoring the treatment of an individual patient.

In order to provide a basis for the diagnosis of a disorder associatedwith expression of UBCLE, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, encoding UBCLE, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withvalues from an experiment in which a known amount of a substantiallypurified polynucleotide is used. Standard values obtained from normalsamples may be compared with values obtained from samples from patientswho are symptomatic for a disorder. Deviation from standard values isused to establish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocolis initiated, hybridization assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding UBCLE may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding UBCLE, or a fragment of a polynucleotide complementary to thepolynculeotide encoding UBCLE, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantitation of closely related DNA or RNA sequences.

Methods which may also be used to quantitate the expression of UBCLEinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and interpolating results from standard curves.(Melby, P. C. et al. (1993) J. Immunol. Methods 159:235-244, and Duplaa,C. et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitationof multiple samples may be accelerated by running the assay in an ELISAformat where the oligomer of interest is presented in various dilutionsand a spectrophotometric or calorimetric response gives rapidquantitation.

In further embodiments, oligonucleotides or longer fragments derivedfrom any of the polynucleotide sequences described herein may be used astargets in a microarray. The microarray can be used to monitor theexpression level of large numbers of genes simultaneously (to produce atranscript image) and to identify genetic variants, mutations, andpolymorphisms. This information may be used in determining genefunction, in understanding the genetic basis of a disorder, indiagnosing a disorder, and in developing and monitoring the activitiesof therapeutic agents.

In one embodiment, the microarray is prepared and used according tomethods known in the art, such as those described in published PCTapplication WO95/11995 (Chee et al.), Lockhart, D. J. et al. (1996; Nat.Biotech. 14:1675-1680), and Schena, M. et al. (1996; Proc. Natl. Acad.Sci. 93:10614-10619).

The microarray is preferably composed of a large number of uniquesingle-stranded nucleic acid sequences, usually either syntheticantisense oligonucleotides or fragments of cDNAs, fixed to a solidsupport. The oligonucleotides are preferably about 6 to 60 nucleotidesin length, more preferably about 15 to 30 nucleotides in length, andmost preferably about 20 to 25 nucleotides in length. For a certain typeof microarray, it may be preferable to use oligonucleotides which areabout 7 to 10 nucleotides in length. The microarray may containoligonucleotides which cover the known 5' or 3' sequence, or may containsequential oligonucleotides which cover the full length sequence orunique oligonucleotides selected from particular areas along the lengthof the sequence. Polynucleotides used in the microarray may beoligonucleotides specific to a gene or genes of interest in which atleast a fragment of the sequence is known or oligonucleotides specificto one or more unidentified cDNAs common to a particular cell or tissuetype or to a normal, developmental, or disease state. In certainsituations, it may be appropriate to use pairs of oligonucleotides on amicroarray. The pairs will be identical, except for one nucleotidepreferably located in the center of the sequence. The secondoligonucleotide in the pair (mismatched by one) serves as a control. Thenumber of oligonucleotide pairs may range from about 2 to 1,000,000.

In order to produce oligonucleotides to a known sequence for amicroarray, the gene of interest is examined using a computer algorithmwhich starts at the 5' end, or, more preferably, at the 3' end of thenucleotide sequence. The algorithm identifies oligomers of definedlength that are unique to the gene, have a GC content within a rangesuitable for hybridization, and lack predicted secondary structure thatmay interfere with hybridization. In one aspect, the oligomers aresynthesized at designated areas on a substrate using a light-directedchemical process. The substrate may be paper, nylon, any other type ofmembrane, filter, chip, glass slide, or any other suitable solidsupport.

In one aspect, the oligonucleotides may be synthesized on the surface ofthe substrate by using a chemical coupling procedure and an ink jetapplication apparatus, such as that described in published PCTapplication WO95/251 116 (Baldeschweiler et al.). In another aspect, agrid array analogous to a dot or slot blot (HYBRIDOT apparatus,GIBCO/BRL) may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system orthermal, UV, mechanical or chemical bonding procedures. In yet anotheraspect, an array may be produced by hand or by using available devices,materials, and machines (including Brinkmann® multichannel pipettors orrobotic instruments), and may contain 8, 24, 96, 384, 1536, or 6144oligonucleotides, or any other multiple from 2 to 1,000,000 which lendsitself to the efficient use of commercially available instrumentation.

In order to conduct sample analysis using the microarrays,polynucleotides are extracted from a biological sample. The biologicalsamples may be obtained from any bodily fluid (blood, urine, saliva,phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissuepreparations. To produce probes, the polynucleotides extracted from thesample are used to produce nucleic acid sequences which arecomplementary to the nucleic acids on the microarray. If the microarrayconsists of cDNAs, antisense RNAs (aRNA) are appropriate probes.Therefore, in one aspect, mRNA is used to produce cDNA which, in turnand in the presence of fluorescent nucleotides, is used to producefragment or oligonucleotide aRNA probes. These fluorescently labeledprobes are incubated with the microarray so that the probe sequenceshybridize to the cDNA oligonucleotides of the microarray. In anotheraspect, nucleic acid sequences used as probes can includepolynucleotides, fragments, and complementary or antisense sequencesproduced using restriction enzymes, PCR technologies, and OLIGOLABELINGor TRANSPROBE kits (Pharmacia & Upjohn) well known in the area ofhybridization technology.

Incubation conditions are adjusted so that hybridization occurs withprecise complementary matches or with various degrees of lesscomplementarity. After removal of nonhybridized probes, a scanner isused to determine the levels and patterns of fluorescence. The scannedimages are examined to determine the degree of complementarity and therelative abundance of each oligonucleotide sequence on the microarray. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large scale correlationstudies or for functional analysis of the sequences, mutations,variants, or polymorphisms among samples. (Heller, R. H. et al. (1997)Proc. Natl. Acad. Sci. 94:2150-2155.)

In another embodiment of the invention, nucleic acid sequences encodingUBCLE may be used to generate hybridization probes useful for mappingthe naturally occurring genomic sequence. The sequences may be mapped toa particular chromosome, to a specific region of a chromosome, or toartificial chromosome constructions, such as human artificialchromosomes (HACs), yeast artificial chromosomes (YACs), bacterialartificial chromosomes (BACs), bacterial P1 constructions, or singlechromosome cDNA libraries, such as those reviewed in Price, C. M. (1993;Blood Rev. 7:127-134) and Trask, B. J. (1991; Trends Genet. 7:149-154).

Fluorescent in situ hybridization (FISH, as described in Verma et al.(1988) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,New York, N.Y.) may be correlated with other physical chromosome mappingtechniques and genetic map data. Examples of genetic map data can befound in various scientific journals or at the Online MendelianInheritance in Man (OMIM) site. Correlation between the location of thegene encoding UBCLE on a physical chromosomal map and a specificdisorder, or predisposition to a specific disorder, may help define theregion of DNA associated with that disorder. The nucleotide sequences ofthe subject invention may be used to detect differences in genesequences between normal, carrier, and affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques, such as linkage analysis using established chromosomalmarkers, may be used for extending genetic maps. Often the placement ofa gene on the chromosome of another mammalian species, such as mouse,may reveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms, or parts thereof, by physical mapping. This providesvaluable information to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, for example, AT to 11q22-23 (Gatti, R. A. etal. (1988) Nature 336:577-580), any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleotide sequence of the subject invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc., among normal, carrier, or affected individuals.

In another embodiment of the invention, UBCLE, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes betweenUBCLE and the agent being tested may be measured.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest as described in published PCT applicationWO84/03564. In this method, large numbers of different small testcompounds are synthesized on a solid substrate, such as plastic pins orsome other surface. The test compounds are reacted with UBCLE, orfragments thereof, and washed. Bound UBCLE is then detected by methodswell known in the art. Purified UBCLE can also be coated directly ontoplates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding UBCLE specificallycompete with a test compound for binding UBCLE. In this manner,antibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with UBCLE.

In additional embodiments, the nucleotide sequences which encode UBCLEmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES

I. ADRETUTo5 cDNA Library Construction

The ADRETUT05 cDNA library was constructed from tumor tissue obtainedfrom the right adrenal gland of a 52 year-old female during a unilateraladrenalectomy. Pathology indicated a pheochromocytoma. Patient historyincluded benign hypertension, depressive disorder, chronic sinusitis,idiopathic proctocolitis, urinary tract infection and irritable colon.Family history included benign hypertension in a sibling,cerebrovascular disease, secondary Parkinsonism and irritable colon inthe mother; atherosclerotic coronary artery disease, hyperlipidemia andmalignant brain neoplasm in siblings and secondary Parkinsonism in thefather.

The frozen tissues were homogenized and lysed in TRIZOL reagent (1 gmtissue/10 ml TRIZOL; Cat. #10296-028; Gibco/BRL), a monophasic solutionof phenol and guanidine isothiocyanate, using a Brinkmann POLYTRONHomogenizer PT-3000 (Brinkmann Instruments, Westbury, N.Y.). After abrief incubation on ice, chloroform was added (1:5 v/v), and the lysatewas centrifuged. The upper chloroform layer was removed to a fresh tubeand the RNA extracted with isopropanol, resuspended in DEPC-treatedwater, and DNase treated for 25 min at 37° C. The RNA was re-extractedonce with acid phenol-chloroform pH 4.7 and precipitated using 0.3Msodium acetate and 2.5 volumes ethanol. The mRNA was then isolated withthe OLIGOTEX kit (QIAGEN, Inc., Chatsworth, Calif.) and used toconstruct the cDNA library.

The mRNA was handled according to the recommended protocols in the (Cat.#18248-013, Gibco/BRL). The cDNAs were fractionated on a SEPHAROSE CL4Bcolumn (Cat. #275105-01; Pharmacia), and those cDNAs exceeding 400 bpwere ligated into pINCY 1. The plasmid pINCY 1 was subsequentlytransformed into DH5a competent cells (Cat. #18258-012; Gibco/BRL).

II Isolation and Sequencing of cDNA Clones

Plasmid DNA was released from the cells and purified using the R.E.A.L.PREP 96 Plasmid Kit (Catalog #26173, QIAGEN). The recommended protocolwas employed except for the following changes: 1) the bacteria werecultured in 1 ml of sterile Terrific Broth (Catalog #2271 1, Gibco/BRL)with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) the cultures wereincubated for 19 hours after the wells were inoculation and then lysedwith 0.3 ml of lysis buffer; 3) following isopropanol precipitation, theDNA pellet was resuspended in 0.1 ml of distilled water. After the laststep in the protocol, samples were transferred to a 96-well block forstorage at 4° C.

The cDNAs were sequenced by the method of Sanger et al. (1975, J. Mol.Biol. 94:441 f), using a Hamilton MICROLAB 2200 (Hamilton, Reno, Nev.)in combination with Peltier thermal cyclers (PTC200 from MJ Research,Watertown, Mass.) and Applied Biosystems 377 DNA sequencing systems.

III. Homology Searching of cDNA Clones and Their Deduced Proteins

The nucleotide sequences and/or amino acid sequences of the SequenceListing were used to query sequences in the GenBank, SwissProt, BLOCKS,and Pima II databases. These databases, which contain previouslyidentified and annotated sequences, were searched for regions ofhomology using BLAST (Basic Local Alignment Search Fool). (Altschul, S.F. (1993) J. Mol. Evol 36:290-300; and Altschul et al. (1990) J. Mol.Biol. 215:403-410.)

BLAST produced alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST was especially useful in determining exact matches orin identifying homologs which may be of prokaryotic (bacterial) oreukaryotic (animal, fungal, or plant) origin. Other algorithms such asthe one described in Smith, T. et al. (1992; Protein Engineering5:35-51), could have been used when dealing with primary sequencepatterns and secondary structure gap penalties. The sequences disclosedin this application have lengths of at least 49 nucleotides and have nomore than 12% uncalled bases (where N is recorded rather than A, C, G,or T).

The BLAST approach searched for matches between a query sequence and adatabase sequence. BLAST evaluated the statistical significance of anymatches found, and reported only those matches that satisfy theuser-selected threshold of significance. In this application, thresholdwas set at 10⁻ 25 for nucleotides and 10⁻¹⁰ for peptides.

Incyte nucleotide sequences were searched against the GenBank databasesfor primate (pri), rodent (rod), and other mammalian sequences (mam),and deduced amino acid sequences from the same clones were then searchedagainst GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp), and eukaryote (eukp), for homology.

IV. Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound. (Sambrook, supra).

Analogous computer techniques applying BLAST are used to search foridentical or related molecules in nucleotide databases such as GenBankor the LIFESEQ™ database (Incyte Pharmaceuticals). This analysis is muchfaster than multiple membrane-based hybridizations. In addition, thesensitivity of the computer search can be modified to determine whetherany particular match is categorized as exact or homologous.

The basis of the search is the product score, which is defined as:##EQU1## The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1% to 2% error, and, with a product score of 70, the match will beexact. Homologous molecules are usually identified by selecting thosewhich show product scores between 15 and 40, although lower scores mayidentify related molecules.

The results of northern analysis are reported as a list of libraries inwhich the transcript encoding UBCLE occurs. Abundance and percentabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a cDNA library, andpercent abundance is abundance divided by the total number of sequencesexamined in the cDNA library.

V. Extension of UBCLE Encoding Polynucleotides

The nucleic acid sequence of Incyte Clone 2501808 was used to designoligonucleotide primers for extending a partial nucleotide sequence tofull length. One primer was synthesized to initiate extension in theantisense direction, and the other was synthesized to extend sequence inthe sense direction. Primers were used to facilitate the extension ofthe known sequence "outward" generating amplicons containing new unknownnucleotide sequence for the region of interest. The initial primers weredesigned from the cDNA using OLIGO 4.06 (National Biosciences), oranother appropriate program, to be about 22 to 30 nucleotides in length,to have a GC content of about 50% or more, and to anneal to the targetsequence at temperatures of about 68° C. to about 72° C. Any stretch ofnucleotides which would result in hairpin structures and primer-primerdimerizations was avoided.

Selected human cDNA libraries (GIBCO/BRL) were used to extend thesequence. If more than one extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

High fidelity amplification was obtained by following the instructionsfor the XL-PCR kit (Perkin Elmer) and thoroughly mixing the enzyme andreaction mix. PCR was performed using the Peltier thermal cycler(PTC200; M. J. Research, Watertown, Mass.), beginning with 40 pmol ofeach primer and the recommended concentrations of all other componentsof the kit, with the following parameters:

    ______________________________________                                        Step 1   94° C. for 1 min (initial denaturation)                         Step 2 65° C. for 1 min                                                Step 3 68° C. for 6 min                                                Step 4 94° C. for 15 sec                                               Step 5 65° C. for 1 min                                                Step 6 68° C. for 7 min                                                Step 7 Repeat steps 4 through 6 for an additional 15 cycles                   Step 8 94° C. for 15 sec                                               Step 9 65° C. for 1 min                                                Step 10 68° C. for 7:15 min                                            Step 11 Repeat steps 8 through 10 for an additional 12 cycles                 Step 12 72° C. for 8 min                                               Step 13 4° C. (and holding)                                          ______________________________________                                    

A 5 μl to 10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6% to 0.8%) agarosemini-gel to determine which reactions were successful in extending thesequence. Bands thought to contain the largest products were excisedfrom the gel, purified using QIAQUICK (QIAGEN), and trimmed of overhangsusing Klenow enzyme to facilitate religation and cloning.

After ethanol precipitation, the products were redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2 to 3 hours, or overnight at 16° C. Competent E. colicells (in 40 μl of appropriate media) were transformed with 3 μl ofligation mixture and cultured in 80 μl of SOC medium. (Sambrook, supra).After incubation for one hour at 37° C., the E. coli mixture was platedon Luria Bertani (LB) agar (Sambrook, supra) containing 2×Carb. Thefollowing day, several colonies were randomly picked from each plate andcultured in 150 μl of liquid LB/2×Carb medium placed in an individualwell of an appropriate commercially-available sterile 96-well microtiterplate. The following day, 5 μl of each overnight culture was transferredinto a non-sterile 96-well plate and, after dilution 1:10 with water, 5μl from each sample was transferred into a PCR array.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction wereadded to each well. Amplification was performed using the followingconditions:

    ______________________________________                                        Step 1   94° C. for 60 sec                                               Step 2 94° C. for 20 sec                                               Step 3 55° C. for 30 sec                                               Step 4 72° C. for 90 sec                                               Step 5 Repeat steps 2 through 4 for an additional 29 cycles                   Step 6 72° C. for 180 sec                                              Step 7 4° C. (and holding)                                           ______________________________________                                    

Aliquots of the PCR reactions were run on agarose gels together withmolecular weight markers. The sizes of the PCR products were compared tothe original partial cDNAs, and appropriate clones were selected,ligated into plasmid, and sequenced.

In like manner, the nucleotide sequence of SEQ ID NO:2 is used to obtain5' regulatory sequences using the procedure above, oligonucleotidesdesigned for 5' extension, and an appropriate genomic library.

VI. Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:2 are employed to screencDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 competent cells (National Biosciences) and labeled bycombining 50 pmol of each oligomer and 250 μCi of [γ-³² P] adenosinetriphosphate (Amersham) and T4 polynucleotide kinase (DuPont NEN®,Boston, Mass.). The labeled oligonucleotides are substantially purifiedusing a SEPHADEX G-25 superfine resin column (Pharmacia & Upjohn). Analiquot containing 10⁷ counts per minute of the labeled probe is used ina typical membrane-based hybridization analysis of human genomic DNAdigested with one of the following endonucleases: Ase I, Bg1 II, Eco RI,Pst I, Xba 1, or Pvu II (DuPont NEN).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (NYTRAN PLUS, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1 ×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT ARautoradiography film (Kodak, Rochester, N.Y.) is exposed to the blots ina, or the blots are place cassette (Molecular Dynamics, Sunnyvale,Calif.) for several hours, hybridization patterns are compared visually.

VII. Microarrays

To produce oligonucleotides for a microarray, one of the nucleotidesequences of the present invention is examined using a computeralgorithm which starts at the 3' end of the nucleotide sequence. Thealgorithm identifies oligomers of defined length that are unique to thegene, have a GC content within a range suitable for hybridization, andlack predicted secondary structure that would interfere withhybridization. The algorithm identifies approximately 20sequence-specific oligonucleotides of 20 nucleotides in length(20-mers). A matched set of oligonucleotides are created in which onenucleotide in the center of each sequence is altered. This process isrepeated for each gene in the microarray, and double sets of twenty20-mers are synthesized and arranged on the surface of the silicon chipusing a light-directed chemical process, such as that described in Chee(supra).

In the alternative, a chemical coupling procedure and an ink jet deviceare used to synthesize oligomers on the surface of a substrate. (SeeBaldeschweiler, supra.) In another alternative, a grid array analogousto a dot or slot blot is used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system orthermal, UV, mechanical, or chemical bonding procedures. A typical arraymay be produced by hand or using available materials and machines andcontain grids of 8 dots, 24 dots, 96 dots, 384 dots, 1536 dots, or 6144dots. After hybridization, the microarray is washed to removenonhybridized probes, and a scanner is used to determine the levels andpatterns of fluorescence. The scanned image is examined to determine thedegree of complementarity and the relative abundance/expression level ofeach oligonucleotide sequence in the microarray.

VIII. Complementary Polynucleotides

Sequences complementary to the UBCLE-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring UBCLE. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO 4.06 primeranalysis software and the coding sequence of UBCLE. To inhibittranscription, a complementary oligonucleotide is designed from the mostunique 5' sequence and used to prevent promoter binding to the codingsequence. To inhibit translation, a complementary oligonucleotide isdesigned to prevent ribosomal binding to the UBCLE-encoding transcript.

IX. Expression of UBCLE

Expression of UBCLE is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector is also used to express UBCLE in E. coli.This vector contains a promoter for β-galactosidase upstream of thecloning site, followed by sequence containing the amino-terminal Met andthe subsequent seven residues of β-galactosidase. Immediately followingthese eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

Induction of an isolated, transformed bacterial strain with isopropylbeta-D-thiogalactopyranoside (IPTG) using standard methods produces afusion protein which consists of the first 8 residues ofβ-galactosidase, about 5 to 15 residues of linker, and the full lengthprotein. The signal residues direct the secretion of UBCLE intobacterial growth media which can be used directly in the following assayfor activity.

X. Demonstration of UBCLE Activity

UBCLE activity is demonstrated by the formation of di-ubiquitinconjugates from free ubiquitin (van Nocker et al. supra). UBCLE isincubated together with 75 pmol ¹²⁵ I-labeled ubiquitin, 20 nM wheat E1,2 mM Mg ATP, 0.1 mM dithiothreitol, and 50 mM Tris-HCl, pH 8.0. Thereaction is incubated for 2 minutes at 4° C. and the di-ubiquitinproduct separated from free ubiquitin by polyacrylamide gelelectrophoresis. Di-ubiquitin is visualized by autoradiography, removedfrom the gel, and counted in a gamma radioisotope counter. The amount ofdi-ubiquitin formed in the reaction is proportional to the activity ofUBCLE in the assay.

XI. Production of UBCLE Specific Antibodies

UBCLE substantially purified using PAGE electrophoresis (Sambrook,supra), or other purification techniques, is used to immunize rabbitsand to produce antibodies using standard protocols. The UBCLE amino acidsequence is analyzed using DNASTAR software (DNASTAR Inc) to determineregions of high immunogenicity, and a corresponding oligopeptide issynthesized and used to raise antibodies by means known to those ofskill in the art. Selection of appropriate epitopes, such as those nearthe C-terminus or in hydrophilic regions, is described by Ausubel(supra) and by others.

Typically, the oligopeptides are 15 residues in length, and aresynthesized using an Applied Biosystems 431A peptide synthesizer usingfmoc-chemistry and coupled to KLH (Sigma, St. Louis, Mo.) by reactionwith N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), following theprocedure described in Ausubel et al., supra. Rabbits are immunized withthe oligopeptide-KLH complex in complete Freund's adjuvant. Resultingantisera are tested for antipeptide activity, for example, by bindingthe peptide to plastic, locking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radio-odinated goat anti-rabbitIgG.

II. Purification of Naturally Occurring UBCLE Using Specific Antibodies

Naturally occurring or recombinant UBCLE is substantially purified byimmunoaffinity chromatography using antibodies specific for UBCLE. Animmunoaffinity column is constructed by covalently coupling UBCLEantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

Media containing UBCLE are passed over the immunoaffinity column, andthe column is washed under conditions that allow the preferentialabsorbance of UBCLE (e.g., high ionic strength buffers in the presenceof detergent). The column is eluted under conditions that disruptantibody/UBCLE binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), andUBCLE is collected.

XIII. Identification of Molecules Which Interact with UBCLE

UBCLE or biologically active fragments thereof are labeled with 1251Bolton-Hunter reagent. (Bolton et al. (1973) Biochem. J. 133:529.)Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled UBCLE, washed, and any wells withlabeled UBCLE complex are assayed. Data obtained using differentconcentrations of UBCLE are used to calculate values for the number,affinity, and association of UBCLE with the candidate molecules.

Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 3                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 197 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: ADRETUT05                                                        (B) CLONE: 2501808                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - -  Met Gln Arg Ala Ser Arg Leu Lys Arg Glu - #Leu His Met Leu Ala        Thr                                                                               1               5 - #                 10 - #                 15             - -  Glu Pro Pro Pro Gly Ile Thr Cys Trp Gln - #Asp Lys Asp Gln Met Asp                   20     - #             25     - #             30                  - -  Asp Leu Arg Ala Gln Ile Leu Gly Gly Ala - #Asn Thr Pro Tyr Glu Lys               35         - #         40         - #         45                      - -  Gly Val Phe Lys Leu Glu Val Ile Ile Pro - #Glu Arg Tyr Pro Phe Glu           50             - #     55             - #     60                          - -  Pro Pro Gln Ile Arg Phe Leu Thr Pro Ile - #Tyr His Pro Asn Ile Asp       65                 - # 70                 - # 75                 - # 80       - -  Ser Ala Gly Arg Ile Cys Leu Asp Val Leu - #Lys Leu Pro Pro Lys Gly                       85 - #                 90 - #                 95              - -  Ala Trp Arg Pro Ser Leu Asn Ile Ala Thr - #Val Leu Thr Ser Ile Gln                   100     - #            105     - #            110                 - -  Leu Leu Met Ser Glu Pro Asn Pro Asp Asp - #Pro Leu Met Ala Asp Ile               115         - #        120         - #        125                     - -  Ser Ser Glu Phe Lys Tyr Asn Lys Pro Ala - #Phe Leu Lys Asn Ala Arg           130             - #    135             - #    140                         - -  Gln Trp Thr Glu Lys His Ala Arg Gln Lys - #Gln Lys Ala Asp Glu Glu       145                 - #150                 - #155                 -         #160                                                                             - -  Glu Met Leu Asp Asn Leu Pro Glu Ala Gly - #Asp Ser Arg Val His        Asn                                                                                              165 - #                170 - #                175            - -  Ser Thr Gln Lys Arg Lys Ala Ser Gln Leu - #Val Gly Ile Glu Lys Lys                   180     - #            185     - #            190                 - -  Phe His Pro Asp Val                                                              195                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 877 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: ADRETUT05                                                        (B) CLONE: 2501808                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - -  CGTTGCTGCG TTGTGAGGGG TGTCAGCTCA GTGCATCCCA GGCAGCTCTT - #AGTGTGGAG    C    60                                                                         - -  AGTGAACTGT GTGTGGTTCC TTCTACTTGG GGATCATGCA GAGAGCTTCG - #CGTCTGAAG    A   120                                                                         - -  GAGAGCTGCA CATGTTAGCC ACAGAGCCAC CCCCAGGCAT CACATGTTGG - #CAAGATAAA    G   180                                                                         - -  ACCAAATGGA TGACCTGCGA GCTCAAATAT TAGGTGGAGC CAACACACCT - #TATGAGAAA    G   240                                                                         - -  GTGTTTTTAA GCTAGAAGTT ATCATTCCTG AGAGGTACCC ATTTGAACCT - #CCTCAGATC    C   300                                                                         - -  GATTTCTCAC TCCAATTTAT CATCCAAACA TTGATTCTGC TGGAAGGATT - #TGTCTGGAT    G   360                                                                         - -  TTCTCAAATT GCCACCAAAA GGTGCTTGGA GACCATCCCT CAACATCGCA - #ACTGTGTTG    A   420                                                                         - -  CCTCTATTCA GCTGCTCATG TCAGAACCCA ACCCTGATGA CCCGCTCATG - #GCTGACATA    T   480                                                                         - -  CCTCAGAATT TAAATATAAT AAGCCAGCCT TCCTCAAGAA TGCCAGACAG - #TGGACAGAG    A   540                                                                         - -  AGCATGCAAG ACAGAAACAA AAGGCTGATG AGGAAGAGAT GCTTGATAAT - #CTACCAGAG    G   600                                                                         - -  CTGGTGACTC CAGAGTACAC AACTCAACAC AGAAAAGGAA GGCCAGTCAG - #CTAGTAGGC    A   660                                                                         - -  TAGAAAAGAA ATTTCATCCT GATGTTTAGG GGACTTGTCC TGGTTCATCT - #TAGTTAATG    T   720                                                                         - -  GTTCTTTGCC AAGGTGATCT AAGTTGCCTA CCTTGAATTT TTTTTTAAAT - #ATATTTGAT    G   780                                                                         - -  ACATAATTTT TGTGTAGTTT ATTTATCTTG TACATATGTA TTTTGAAATC - #TTTTAAACC    T   840                                                                         - -  GAAAAATAAA TAGTCATTTA ATGTTGAAAA AAAAAAA     - #                       - #     877                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 147 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: 1145691                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - -  Met Ala Leu Lys Arg Ile Asn Lys Glu Leu - #Ser Asp Leu Ala Arg        Asp                                                                               1               5 - #                 10 - #                 15             - -  Pro Pro Ala Gln Cys Ser Ala Gly Pro Val - #Gly Asp Asp Met Phe His                   20     - #             25     - #             30                  - -  Trp Gln Ala Thr Ile Met Gly Pro Asn Asp - #Ser Pro Tyr Gln Gly Gly               35         - #         40         - #         45                      - -  Val Phe Phe Leu Thr Ile His Phe Pro Thr - #Asp Tyr Pro Phe Lys Pro           50             - #     55             - #     60                          - -  Pro Lys Val Ala Phe Thr Thr Arg Ile Tyr - #His Pro Asn Ile Asn Ser       65                 - # 70                 - # 75                 - # 80       - -  Asn Gly Ser Ile Cys Leu Asp Ile Leu Arg - #Ser Gln Trp Ser Pro Ala                       85 - #                 90 - #                 95              - -  Leu Thr Ile Ser Lys Val Leu Leu Ser Ile - #Cys Ser Leu Leu Cys Asp                   100     - #            105     - #            110                 - -  Pro Asn Pro Asp Asp Pro Leu Val Pro Glu - #Ile Ala Arg Ile Tyr Lys               115         - #        120         - #        125                     - -  Thr Asp Arg Asp Lys Tyr Asn Arg Ile Ser - #Arg Glu Trp Thr Gln Lys           130             - #    135             - #    140                         - -  Tyr Ala Met                                                              145                                                                         __________________________________________________________________________

What is claimed is:
 1. An isolated and purified polynucleotide encodinga polypeptide comprising the amino acid sequence of SEQ ID NO:1.
 2. Acomposition comprising the polynucleotide of claim
 1. 3. An isolated andpurified polynucleotide which is completely complementary to thepolynucleotide of claim
 1. 4. An isolated and purified polynucleotidecomprising SEQ ID NO:2.
 5. An isolated and purified polynucleotide whichis completely complementary to the polynucleotide of claim
 4. 6. Anexpression vector comprising the polynucleotide of claim
 1. 7. A hostcell comprising the expression vector of claim
 6. 8. A method forproducing a polypeptide comprising the amino acid sequence of SEQ IDNO:1, the method comprising the steps of:(a) culturing the host cell ofclaim 7 under conditions suitable for the expression of the polypeptide;and (b) recovering the polypeptide from the host cell culture.
 9. Amethod for detecting a polynucleotide encoding UBCLE in a biologicalsample containing nucleic acids, the method comprising the steps of:(a)hybridizing the polynucleotide of claim 3 to at least one of the nucleicacids of the biological sample, thereby forming a hybridization complex;and (b) detecting the hybridization complex, wherein the presence of thehybridization complex correlates with the presence of a polynucleotideencoding UBCLE in the biological sample.
 10. The method of claim 9wherein the nucleic acids of the biological sample are amplified by thepolymerase chain reaction prior to the hybridizing step.